APOBEC3 (A3) proteins are members of an innate immune response that provide a defense against HIV-1 and other pathogens. In the absence of the HIV-1 protein Vif (Vif1), the A3 proteins are incorporated into virions in the virus producer cells and inhibit viral replication by deaminating cytidines in the minus-strand of viral DNA during reverse transcription in the target cells, resulting in extensive G-to-A hypermutation of the viral genome. In addition to inactivating most of the viral genomes through lethal hypermutation, we and others have shown that A3G and A3F also inhibit viral DNA synthesis and integration. To overcome these host defenses, Vif1 binds to the A3 proteins and targets them for proteasomal degradation, preventing their incorporation into virions. Defining the interactions of Vif1 with A3G and A3F at the molecular level could provide two potential targets for the development of antiviral drugs to suppress A3G and A3F degradation. Our goal is to understand the structure and function of Vif and A3 proteins. We will gain insights into the structures of Vif:A3 complexes through mutational and comparative analyses and generate reagents for structural studies. ___Strategies to control HIV-1 replication without antiviral therapy are needed to achieve a functional cure. To exploit the innate antiviral function of A3G, we developed novel self-activating lentiviral vectors that efficiently deliver an HIV-1 Vif-resistant A3G-D128K mutant to target cells. To circumvent A3G expression in virus-producing cells, which diminishes virus production, a vector containing two overlapping fragments of A3G-D128K was designed that maintained the gene in an inactive form in the virus-producer cells. However, during transduction of target cells, homologous recombination between the direct repeats reconstituted an active A3G-D128K in 88-98% of transduced cells. Feasibility of human gene therapy was supported by 30% transduction of CD34+ hematopoietic stem and progenitor cells. A3G-D128K expression in T-cell lines CEM, CEMSS, and PM1 potently inhibited spreading infection of HIV-1 subtypes by C-to-U deamination, leading to lethal G-to-A hypermutation and inhibition of reverse transcription. A3G-D128K expression in CEM cells potently suppressed HIV-1 replication for 3.5 months without emergence of detectable resistant virus, suggesting a high genetic barrier for evolution of A3G-D128K resistance. These studies provide a proof of principle that A3G-D128K gene therapy is potentially a viable strategy to achieve a functional cure for HIV-1. ___Recent studies have shown that HIV-1 Vif interacts with host factor CBFbeta and that this interaction is critical for Vif-mediated degradation of A3 proteins. It was thought that the Vif-CBFbeta interaction increases the stability of Vif, which facilitates its interactions with cullin5-RBX2-ubiquitin ligase complex that are needed for inducing degradation of A3 proteins. In collaboration with Yong Xiong (Yale University), we determined the structure of a complex of Vif, CBFbeta, and A3F C-terminal domain, and unexpectedly found that A3F directly interacts with CBFbeta. The in vivo significance of the A3F-CBFbeta interaction was established by showing that mutations in CBFbeta prevent Vif-mediated degradation of A3F and that compensatory mutations that restore the interactions also restore A3F degradation. ___In addition to HIV-1 group M, the primary HIV-1 group responsible for the AIDS epidemic, HIV-1 groups N, O, and P have been shown to infect humans on rare occasions. We have characterized the Vifs from these four groups and found that some groups can induce degradation of A3G mutant D128K, which is resistant to degradation by group M Vif. Through mutational analyses, we have identified mutations in the N-terminal region of group M Vif that confer partial ability to induce degradation of the D128K mutant of A3G. These studies have helped to define the interactions between Vif and A3G that are critical for Vif's ability to overcome the A3G restriction. ___In collaboration with Hiroshi Matsuo (Leidos Biomedical Research, Inc., Frederick National Laboratory), we determined the structure of the C-terminal catalytic domain (CTD) of A3G in complex with a single-strand DNA (ssDNA) substrate. To overcome weak DNA-binding affinity between A3G and the CTD, we generated a catalytically active variant of A3G-CTD that binds ssDNA stronger than wild type. This A3G-CTD variant was co-crystallized with a 9-nucleotide ssDNA containing a 5'-TCCCA target sequence with all 9 nucleotides well resolved in the structure. The nucleotides within the 5'-TCCCA target sequence show numerous interactions with A3G-CTD, explaining the nucleotide specificity preferences. Furthermore, the backbone architecture of the protein changed upon ssDNA binding, enabling the target sequence to fit. These results provide fundamental insights into the mechanisms by which A3 proteins recognize their specific substrate sequences. ___Somatic mutations generated by A3B are common in many human cancers, but their burden varies within and between cancer types. In collaboration with Ludmila Prokunina-Olsson (Laboratory of Translational Genomics, NCI), we showed that alternative splicing of A3B (A3B) results in reduced expression of mutagenic A3B, leading to decreased A3B signature mutations. Importantly, we showed that A3B exon 5 splicing can be modulated by SF3B1 pladienolide B inhibitor leading to reduced mutagenic A3B1 protein levels. We propose that pladienolide B-based drugs may hold promise to modulate A3-mediated mutagenesis in human cancers.